Fluorescence Of Human Serum Albumin Research Articles

Binding mechanism of trans-N-caffeoyltyramine and human serum albumin: Investigation by multi-spectroscopy and docking simulation

trans-N-Caffeoyltyramine (TNC), which was isolated from the Cortex Lycii in our laboratory, is a phenolic amide compound with multiple pharmacological activities. The interaction between TNC and human serum albumin (HSA) was studied by Nuclear magnetic resonance (NMR) relaxation experiment, fluorescence spectroscopy, and docking simulation. NMR methodology is based on the analysis of selective and non-selective spin-lattice relaxation rate enhancements of TNC protons in the presence of the HSA. Result indicated that the interaction occurred between HSA and TNC, and changed the proton magnetic environment of TNC. Fluorescence spectroscopy confirmed that TNC displayed a strong capability to quench the fluorescence of HSA, and the acting forces for binding were hydrogen bonds and van der Waals forces. Furthermore, the circular dichroism, synchronous, and three-dimensional fluorescence spectra, which were employed to determine the conformation of protein, revealed that binding of TNC with HSA could induce conformational changes in HSA. In addition, the molecular modeling results exhibited that TNC mainly bonded to site I in sub-domain IIA of HSA.

Biological Activity of Japanese Quince Extract and Its Interactions with Lipids, Erythrocyte Membrane, and Human Albumin.

The aim of the study was to determine in vitro biological activity of fruit ethanol extract from Chaenomeles speciosa (Sweet) Nakai (Japanese quince, JQ) and its important constituents (−)-epicatechin (EC) and chlorogenic acid (CA). The study also investigated the structural changes in phosphatidylcholine (PC) liposomes, dipalmitoylphosphatidylcholine liposomes, and erythrocyte membranes (RBC) induced by the extract. It was found that the extract effectively inhibits oxidation of RBC, induced by 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), and PC liposomes, induced by UVB radiation and AAPH. Furthermore, JQ extract to a significant degree inhibited the activity of the enzymes COX-1 and COX-2, involved in inflammatory reactions. The extract has more than 2 times greater activity in relation to COX-2 than COX-1 (selectivity ratio 0.48). JQ extract stimulated growth of the beneficial intestinal bacteria Lactobacillus casei and Lactobacillus plantarum. In the fluorimetric method by means of the probes Laurdan, DPH and TMA-DPH, and 1H-NMR, we examined the structural changes induced by JQ and its EC and CA components. The results show that JQ and its components induce a considerable increase of the packing order of the polar heads of lipids with a slight decrease in mobility of the acyl chains. Lipid membrane rigidification could hinder the diffusion of free radicals, resulting in inhibition of oxidative damage induced by physicochemical agents. JQ extract has the ability to quench the intrinsic fluorescence of human serum albumin through static quenching. This report thus could be of huge significance in the food industry, pharmacology, and clinical medicine.

Probing the interactions of bromchlorbuterol-HCl and phenylethanolamine A with HSA by multi-spectroscopic and molecular docking technique

Using fluorescence quenching, fluorescence lifetime, (UV+vis) absorption, circular dichroism (CD) and molecular docking technique, the interactions of human serum albumin (HSA) with bromchlorbuterol-HCl (BCB) and phenylethanolamine A (PEA) were investigated. The quenching rate constants and binding constants for BCB/PEA with HSA were determined at T=(292.15, 302.15 and 312.15)K respectively, which were all decreased with the increase of the temperature, showing not a dynamic quenching. The fluorescence lifetime of HSA with BCB/PEA had changed little compared to that of HSA alone (τ0), further confirming that BCB/PEA quenching of intrinsic fluorescence of HSA is a static quenching. The effects of K+, Ca2+, Cu2+, Zn2+ and Fe3+ on the binding were studied. The analysis of the thermodynamic parameters for BCB/(PEA+HSA) showed that BCB/PEA could bind to HSA via hydrophobic force. The binding distances were determined as 2.90 and 4.11nm for (BCB+HSA) and (PEA+HSA) based on the Förster’s non-radiative energy transfer theory (FRET). Synchronous fluorescence and CD spectra indicated that the conformation of HSA was changed by BCB/PEA. The competitive studies for the drug with site marker suggested that both BCB and PEA were bound at Sudlow’s sites I (sub-domain IIA, also known as indometacin binding site) in HSA, and the results of the study of molecular docking also leads to the same conclusion. The competitive binding experiments for the two drugs were also performed, which further indicates that PEA and BCB could share the same binding site, and PEA has a much stronger binding capacity than BCB.

Biophysical and Molecular Investigation of the Interaction between Enterolactone and Human Serum Albumin

Plant polyphenol lignans are naturally occurring phenolic compounds broadly available in cereals, legumes, fruits, vegetables, and whole grains. Enterolactone (ENL), a major metabolite of plant-based lignans, is shown to inhibit the proliferation and development of various cancers. Moreover, human serum albumin (HSA) is the most abundant protein in blood plasma and serves as a carrier of various drugs. In this study for the first time, the interactions between ENT and HSA was investigated by fluorescence and circular dichroism (CD) spectroscopic methods under physiological conditions (pH=7.4) and also molecular docking. Results demonstrated that the intrinsic fluorescence of HSA was remarkably quenched by ENT with binding constant of 1.6×109 M-1 at 298.15K. The thermodynamic parameters showed that both hydrophobic interactions and hydrogen bonds play the main role in the formation of ENT-HSA complex and also the binding process was spontaneous (ΔG=-12.5 kcal mol-1). The percentage of α-helices in HSA increased dramatically from 57% to 84% with increasing the ENT concentration according to CD spectroscopic results when the drug to protein molar ratio was 1.3. Molecular docking confirmed that interaction of ENT with the hydrophobic pocket of HSA, close to the Trp214 residue, through hydrophobic interaction and hydrogen bonds in agreement with the experimental data. Our results suggested that enterolactone can be efficiently stored by HSA due to the relatively high value of Kb. Furthermore, HSA could be introduced as a suitable carrier for drug delivery of ENT.

Binding between Saikosaponin C and Human Serum Albumin by Fluorescence Spectroscopy and Molecular Docking.

Saikosaponin C (SSC) is one of the major active constituents of dried Radix bupleuri root (Chaihu in Chinese) that has been widely used in China to treat a variety of conditions, such as liver disease, for many centuries. The binding of SSC to human serum albumin (HSA) was explored by fluorescence, circular dichroism (CD), UV-vis spectrophotometry, and molecular docking to understand both the pharmacology and the basis of the clinical use of SSC/Chaihu. SSC produced a concentration-dependent quenching effect on the intrinsic fluorescence of HSA, accompanied by a blue shift in the fluorescence spectra. The Stern-Volmer equation showed that this quenching was dominated by static quenching. The binding constant of SSC with HSA was 3.72 × 103 and 2.99 × 103 L·mol−1 at 26 °C and 36 °C, respectively, with a single binding site on each SSC and HSA molecule. Site competitive experiments demonstrated that SSC bound to site I (subdomain IIA) and site II (subdomain IIIA) in HSA. Analysis of thermodynamic parameters indicated that hydrogen bonding and van der Waals forces were mostly responsible for SSC-HSA association. The energy transfer efficiency and binding distance between SSC and HSA was calculated to be 0.23 J and 2.61 nm at 26 °C, respectively. Synchronous fluorescence and CD measurements indicated that SSC affected HSA conformation in the SSC-HSA complex. Molecular docking supported the experimental findings in conformational changes, binding sites and binding forces, and revealed binding of SSC at the interface between subdomains IIA-IIB.

Synthesis, structure, and biological evaluation of a copper(ii) complex with fleroxacin and 1,10-phenanthroline.

A novel mixed-ligand Cu(ii) complex combined with the quinolone drug fleroxacin and 1,10-phenanthroline was synthesized in this work. The crystal structure of the complex was characterized via X-ray crystallography, which was the first reported single crystal complex of fleroxacin. Results showed that Cu(ii) was coordinated through pyridone oxygen and one carboxylate oxygen atom of fleroxacin, as well as two nitrogen atoms from 1,10-phenanthroline. Various characterization methods, including Fourier transform infrared, elementary analysis, thermogravimetry, and X-ray powder diffraction, were applied. The Cu(ii)-quinolone complex exhibited favorable biological activities, and was proved to be capable of transforming supercoiled PUC19 DNA into nicked form under hydrolytic conditions. The obtained pseudo-Michaelis-Menten kinetic parameter was 12.64 h(-1), which corresponded to a million-fold rate enhancement in DNA cleavage. In addition, the interaction capacity of the complex with human serum albumin (HSA) was investigated. The results demonstrated a moderately intense combination between HSA and the complex. The complex evidently quenched the fluorescence of HSA. Approximately 19.2% of the quenching was attributed to Förster resonance energy transfer (FRET), whereas the rest was caused by ground-state complex formation (molar ratio of HSA : complex = 1 : 2). The energy of the complex was excited during FRET, which increased the fluorescence of the complex by approximately 18%.

Synthesis, characterization, in vitro cytotoxicity, in silico ADMET analysis and interaction studies of 5-dithiocarbamato-1,3,4-thiadiazole-2-thiol and its zinc(ii) complex with human serum albumin: combined spectroscopy and molecular docking investigations

The binding site of new complex Zn(ii) of 5-dithiocarbamato-1,3,4-thiadiazole-2-thiol and HAS.

Interactions of human serum albumin with bioactive 3H-imidazo[4,5-a]acridines: Insights from fluorescence spectroscopic studies

Several 3H-imidazo[4,5-a]acridine derivatives were conveniently synthesized by the reaction of imidazo[4,5-a]acridones in boiling POCl3. The imidazoacridones were obtained by rearrangement of 3H-imidazo[4',5':3,4]benzo[c]isoxazoles in concentrated sulfuric acid containing nitrous acid at room temperature. The structures of all newly synthesized compounds were confirmed by IR, 1H NMR, and mass spectral data. The interactions of 3H-imidazo[4,5-a]acridines with human serum albumin (HSA) were studied by fluorescence spectroscopy. The binding of 3H-imidazo[4,5-a]acridines quenches the HSA fluorescence, revealing a 1: 1 interaction with a binding constant of about 2.34 × 105–3.16 × 106 M–1. A decrease in fluorescence intensity at 339 nm, when excited at 280 nm, is attributed to changes in the environment of the protein fluorophores caused by the presence of the ligand. The differences in interactions of 3H-imidazo[4,5-a]acridines with HSA were observed using spectrofluorimetry technique.

Human Serum Albumin Interactions with Bioactive 3H-Imidazo[4,5-A]Acridin-11(6H)-Ones Studied by Fluorescence Spectroscopy

The key intermediates 3H-imidazo[4′,5′:3,4]benzo[c]isoxazoles were synthesized by the reaction of N-alkyl-5-nitrobenzimidazoles with aryl acetonitriles under basic conditions. These compounds were converted to 3H-imidazo[4,5-a]acridin-11(6H)-ones by the Tanasescu reaction in excellent yields. The structures of all newly synthesized compounds were confirmed by IR, 1H NMR, and mass spectroscopic data. The interactions of 3H-imidazo[4,5-a]acridin-11-ones with human serum albumin (HSA) have been studied by fluorescence spectroscopy. The binding of 3H-imidazo[4,5-a]acridin-11(6H)-ones quenches the HSA fluorescence, revealing a 1 : 1 interaction with a binding constant of about 4.50 × 104 – 1.25 × 105 M-1. A decrease in the fluorescence intensity at 339.41 nm, when excited at 280 nm, is attributed to changes in the environment of protein fluorophores caused by the presence of the ligands. Differences in the interactions of 3H-imidazo[4,5-a]acridin-11(6H)-one derivatives with HSA were observed using the spectrofluorimetry technique. Thermodynamic characteristics and the parameters of HSA binding with these compounds are in accordance with the antibacterial activity of various derivatives of 3H-imidazo[4,5-a]acridin-11(6H)-one.

Interaction of diuron to human serum albumin: Insights from spectroscopic and molecular docking studies

ABSTRACTThis investigation was undertaken to determine the interaction of diuron with human serum albumin (HSA) was studied by monitoring the spectral behavior of diuron-HSA system. The fluorescence of HSA at 340 nm excited at 230 nm was obviously quenched by diuron due to dynamic collision and the quenching constant was of the order of 104 L mol−1 at 310 K. However, no fluorescence quenching was observed when excited at 280 nm. Thermodynamic investigations revealed that the combination between diuron and HSA was entropy driven by predominantly hydrophobic interactions. The binding of diuron induced the drastic reduction in α-helix conformation and the significant enhancement in β-turn conformation of HSA. In addition, both sites marker competition study and molecular modeling simulation evidenced the binding of diuron to HSA primarily took place in subdomain IIIA (Sudlow's site II).

Automated evaluation of protein binding affinity of anti-inflammatory choline based ionic liquids

In this work, an automated system for the study of the interaction of drugs with human serum albumin (HSA) was developed. The methodology was based on the quenching of the intrinsic fluorescence of HSA by binding of the drug to one of its binding sites. The fluorescence quenching assay was implemented in a sequential injection analysis (SIA) system and the optimized assay was applied to ionic liquids based on the association of non-steroidal anti-inflammatory drugs with choline (IL-API).In each cycle, 100µL of HSA and 100µL of IL-API (variable concentration) were aspirated at a flow rate of 1mLmin−1 and then sent through the reaction coil to the detector where the fluorescence intensity was measured.In the optimized conditions the effect of increasing concentrations of choline ketoprofenate and choline naproxenate (and respective starting materials: ketoprofen and naproxen) on the intrinsic fluorescence of HSA was studied and the dissociation constants (Kd) were calculated by means of models of drug–protein binding in the equilibrium. The calculated Kd showed that all the compounds bind strongly to HSA (Kd<100µmolL−1) and that the use of the drugs in the IL format does not affect or can even improve their HSA binding.The obtained results were compared with those provided by a conventional batch assay and the relative errors were lower than 4.5%. The developed SIA methodology showed to be robust and exhibited good repeatability in all the assay conditions (rsd<6.5%).

Affinity of a mononuclear monofunctional anticancer Pt(II) complex to human serum albumin: a spectroscopic approach.

The interaction of a mononuclear monofunctional anticancer Pt(II) complex, [PtLCl]Cl (L = 4′-bis(pyridine-2-ylmethyl)amino-2-phenylbenzothiazole) (1), and human serum albumin (HSA) was investigated under physiological conditions using UV–Vis absorption, circular dichroism, fluorescence, and synchronous fluorescence. The experimental results suggested that the Pt(II) complex could bind to HSA, induce conformation and microenvironmental changes of HSA with a moderate binding affinity, and quench the intrinsic fluorescence of HSA through a static quenching mechanism. The thermodynamic parameters, ΔG°, ΔH°, and ΔS°, calculated at different temperatures, indicated that the binding reaction was spontaneous and hydrophobic forces and π–π stacking played major roles in the association. Based on the number of binding sites, it was considered that one molecule of complex 1 could bind to a single site of HSA. In view of the results of site marker competition experiments, the reactive site of HSA to complex 1 mainly located in subdomain IIA (site I). Moreover, the binding distance, r, between donor (HSA) and acceptor (complex 1) was 4.69 nm according to Förster nonradiation energy transfer theory. The present study provides relevant and useful information that can be used for the design and application of mononuclear monofuctional Pt(II) complexes in biomedical sciences.

Interaction of a novel starch‐capped CdS quantum dots with human serum albumin and bovine serum albumin

A novel CdS quantum dots (QDs) were successfully synthesized in aqueous phase using poly (acrylic acid) grafted onto starch (starch‐g‐PAA). UV–vis spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM), and Fourier transform infrared (FT‐IR) techniques proved that the prepared starch‐capped CdS QDs are composed of a CdS core and a polymeric shell with homogeneous size distributions. In order to study its biological behavior, multi‐spectroscopic techniques were adopted to investigate their conjugation with human serum albumin (HSA) and bovine serum albumin (BSA) proteins. The recorded UV–vis spectra, cyclic voltammetry (CV) analysis, and the calculated rate of HSA and BSA fluorescence quenching by the QDs demonstrated that the interaction between them leads to the formation of QDs‐HSA and QDs‐BSA complexes. Moreover, FT‐IR spectra showed the conformational changes of the protein in the presence of obtained QDs, indicating the presence of interaction between modified QDs and proteins.

Spectroscopic study on flavonoid–drug interactions: Competitive binding for human serum albumin between three flavonoid compounds and ticagrelor, a new antiplatelet drug

The effects of three kinds of flavonoids, quercetin, rutin and baicalin, on the binding of ticagrelor to human serum albumin (HSA) were systematically investigated using fluorescence, UV–vis absorption and circular dichroism (CD) spectroscopic techniques. The results indicated that ticagrelor strongly quenched the HSA fluorescence by the style of static quenching and non-radiation energy transferring as a result of the HSA–ticagrelor complex formation, while the presence of flavonoids could not change the quenching mechanism. Ticagrelor could spontaneously bind in site I on HSA with high affinity, and this binding process was mainly driven by both hydrophobic forces and hydrogen bonding. The significantly decreased binding affinity and the unchanged binding mode and distance between ticagrelor and HSA indicated that that flavonoids could compete against ticagrelor in site I, and baicalin was the most effective competitor. The conformation investigation of HSA further confirmed the flavonoid/ticagrelor competitive binding mechanism.

Investigations on the effects of Cu(2+) on the structure and function of human serum albumin.

Human serum albumin (HSA) is the most prominent protein in blood plasma with important physiological functions. Although copper is an essential metal for all organisms, the massive utilization of copper has led to concerns regarding its potential health impact. To better understand the potential toxicity and toxic mechanisms of Cu(2+), it is of vital importance to characterize the interaction of Cu(2+) with HSA. The effect of Cu(2+) on the structure and function of HSA in vitro were investigated by biophysical methods including fluorescence techniques, circular dichroism (CD), time-resolved measurements, isothermal titration calorimetry (ITC), molecular simulations and esterase activity assay. Multi-spectroscopic measurements proved that Cu(2+) quenched the intrinsic fluorescence of HSA in a dynamic process accompanied by the formation of complex and alteration of secondary structure. But the Cu(2+) had minimal effect on the backbone and secondary structure of HSA at relatively low concentrations. The ITC results indicated Cu(2+) interacted with HSA spontaneously through hydrophobic forces with approximately 1 thermodynamic identical binding sites at 298 K. The esterase activity of HSA was inhibited obviously at the concentration of 8 × 10(-5) M. However, molecular simulation showed that Cu(2+) mainly interacted with the amino acid residues Asp (451) by the electrostatic force. Thus, we speculated the interaction between Cu(2+) and HSA might induce microenvironment of the active site (Arg 410). This study has provided a novel idea to explore the biological toxicity of Cu(2+) at the molecular level.

Multispectroscopic Investigation of the Interaction Between two Ruthenium(II) Arene Complexes of Curcumin Analogs and Human Serum Albumin.

The interaction between two ruthenium(II) arene complexes of curcumin analogs and human serum albumin (HSA) was systematically investigated by multispectroscopic techniques. The fluorescence spectral results indicated that two complexes quenched the intrinsic fluorescence of HSA through static quenching mode. The quenching constants and the corresponding thermodynamic parameters at different temperatures were calculated. The binding interactions of two complexes with HSA resulted in the complex formation of complex-HSA, and the van der Waals interactions and hydrogen bond interactions played major roles in the complex stabilization. The distances between HSA and two complexes were obtained according to fluorescence resonance energy transfer theory. The site competitive replacement experiments illustrated that two complexes mainly bounded with HSA on site I. The results of synchronous fluorescence spectra, three-dimensional fluorescence spectra, FT-IR spectra, and circular dichroism spectra indicated that the secondary structure of HSA was changed at the present of two complexes. The results of mass spectrometry further validated the binding interaction and the binding number between two complexes and HSA.

Study on the interactions of mapenterol with serum albumins using multi-spectroscopy and molecular docking.

The interactions of mapenterol with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated systematically using fluorescence spectroscopy, absorption spectroscopy, circular dichroism (CD) and molecular docking techniques. Mapenterol has a strong ability to quench the intrinsic fluorescence of BSA and HSA through static quenching procedures. At 291 K, the binding constants, Ka, were 1.93 × 10(3) and 2.73 × 10(3) L/mol for mapenterol-BSA and mapenterol-HAS, respectively. Electrostatic forces and hydrophobic interactions played important roles in stabilizing the mapenterol-BSA/has complex. Using site marker competitive studies, mapenterol was found to bind at Sudlow site I on BSA/HSA. There was little effect of K(+), Ca(2+), Cu(2+), Zn(2+) and Fe(3+) on the binding. The conformation of BSA/HSA was changed by mapenterol, as seen from the synchronous fluorescence spectra. The CD spectra showed that the binding of mapenterol to BSA/HSA changed the secondary structure of BSA/HSA. Molecular docking further confirmed that mapenterol could bind to Sudlow site I of BSA/HSA. According to Förster non-radiative energy transfer theory (FRET), the distances r0 between the donor and acceptor were calculated as 3.18 and 2.75 nm for mapenterol-BSA and mapenterol-HAS, respectively.

Biophysical characterization of the interaction between human serum albumin and n-dodecyl β-d-maltoside: A multi-technique approach

We have studied the effect of biocompatible sugar based surfactant n-dodecyl β-d-maltoside (DDM) on the conformation of human serum albumin (HSA). A multi-technique approach was applied in order to understand the type of interaction and effect of DDM on the secondary and tertiary structure of HSA. Surface tension measurement showed that HSA shifted the critical micelle concentration (cmc) of the surfactant to the higher side that clarifies the complex formation between DDM and HSA which was also confirmed by UV absorption spectroscopy. Fluorescence quenching measurements showed that fluorescence of HSA was quenched by the addition of DDM with a prominent blue shift indicative of the involvement of hydrophobic interaction which was further confirmed by extrinsic fluorescence of organic dye 1-anilino-8-naphthalenesulfonate. Synchronous fluorescence measurement trends suggested that the hydrophobicity increases near the tryptophan residue while an increase in the polarity was observed near tyrosine residues. A collective information obtained by circular dicroism (CD) and Fourier-transform infra-red (FTIR) spectroscopies along with dynamic light scattering revealed the partial unfolding of the protein.

CHARACTERIZATION OF THE INTERACTION OF CdTe QUANTUM DOTS WITH HUMAN SERUM ALBUMIN BY OPTICAL SPECTROSCOPIC TECHNIQUES

The interaction between CdTe quantum dots (QDs) and human serum albumin (HSA) was studied by absorption and photoluminescence spectroscopy. Three aqueous-compatible samples of colloidal CdTe nanoparticles with average sіze of 2.8, 2.9 and 3.1 nm were tested. In the absorption spectra of the colloidal CdTe QDs exciton band was found to be shifted to higher photon energy as compared with that for bulk crystals due to the quantum confinement effect. It was shown that addition of HSA to colloidal CdTe nanoparticles leds to a gradual decrease of absorption and broadening of exciton structure. The photoluminescence quenching results indicated that the quenching effect of QDs on HSA fluorescence depend on the size and temperature and the nature of quenching is static, resulting in forming QDs–HSA complexes. Stern – Volmer plots were made and quenching constants were thus obtained. The results suggested the quenching constants increasing with increasing of the sizes QDs and decrease with increasing temperatures of QDs-HSA solutions.

Investigation of the Binding Behavior between the S-heterocyclic Aromatic Palladium(II) Complex and Human Serum Albumin: Spectroscopic Approach

The interaction of 1, 10-phenanthroline octhyldithiocarbamato palladium(II) nitrate ([Pd(Oct-dtc)(phen)]NO3) with human serum albumin (HSA) has been investigated by various spectroscopic techniques under physiological conditions. Here, HSA was titrated with the Pd(II) complex, followed by UV–Vis absorption spectroscopy to estimate a binding constant (Kb) and other thermodynamic parameters. The results indicate that the Pd (II) complex has a high affinity for bind HSA. Thermodynamic analysis showed that the enthalpy (ΔH°) and entropy changes (ΔS°) are positive and Gibbs free energy change (ΔG°) is negative which indicated that hydrophobic interactions played the predominant role in the binding process. Fluorescence spectroscopy were used to show the mechanism and binding parameters of this interaction. Utilizing the Stern–Volmer equation, the Pd(II) complex quenched the intrinsic fluorescence of HSA via a static quenching procedure. The specific binding distances between the tryptophan (donor) proteins and Pd(II) complex (acceptor) were estimated by Forster resonance energy transfer. The CD results also showed the conformational changes on serum albumin upon binding with the Pd(II) complex.